(1) Field of the Invention
The present invention relates to a synthetically derived peptide which is believed to be structurally related to a precursor peptide to a bacteriocin, preferably bacteriocin LL-2 produced by Lactococcus lactis subspecies lactis NRRL-B-18809. The peptide is referred to hereinafter as a "precursor peptide". In particular the present invention relates to a precursor peptide which is active against Listeria monocytogenes and inactive against Lactococcal food fermenting strains so that the precursor peptide is useful in fermented foods.
(2) Description of Related Art
Nisin and LL-2 are bacteriocins which differ from each other by a single amino acid in position 27. Nisin has a histidine (His) residue and LL-2 has an asparagine (Asn) residue. The compounds are characterized by a lanthionine bridge between residues 3 and 7.
In the preparation of precursor peptides, one approach is to chemically or thermally inactivate the pathway in the bacterium responsible for protein modification and then hope that the immunity genes also recognize the unmodified "immature" form of the peptide. The precursor peptide gene product would be self-lethal to the bacterium unless there is such immunity. This approach is time consuming to complete and may result in the intended modification but without a method for production due to lack of immunity. The applicability of this strategy is also less than certain, since the inactivation of a multi-enzyme pathway would be required, which might be difficult to obtain without losing viability of the bacterium due to a high level of mutations throughout the genome. Also, one or more of the enzymes responsible for post-translational modification of the precursor peptide to produce the bacteriocin might also be essential to cell viability. Further a lack of information exists regarding the generality of side chain dehydration and lanthionine formation in cellular proteins. Also, the bacterial strain might have developed specialized transport mechanisms which serve to keep unmodified bacteriocin inside and modified bacteriocin outside of the cell. To maintain a system of modification enzymes and then allow unmodified bacteriocin precursor peptide to escape the confines of the cell is contradictory.
A second approach is to clone the DNA pre-protein sequence into a foreign bacterium lacking the enzymes for post-translational modification and to induce expression. Quantities of pre-protein could be obtained by purification of the growth medium. Again, the same problems with immunity and transport in the modified bacterial strains may apply. Although this alternative is attractive, a much greater effort is required with a much less chance at successfully isolating the desired product.
Keeping in mind the difficulty of producing a bacteriocin precursor peptide by microbiological methods, undertaking of such a project would require the expectation that study of the precursor peptide would be interesting in some way. If the prediction were made that the immature peptide is active, one would reasonably ask why does the bacterium take on the energy cost to modify an already functional peptide. Organisms keep notably conservative energy budgets and do not generally invest energy in frivolous protein modification. The possibility exists that modifications evolved in order to provide an evolutionary advantage such as to prolong the lifetime of the active state by evading degrading enzymes or to maintain activity in a reducing or dehydrating environment. Lanthionine bridging is stable under many conditions which disrupt disulfide bridging. However, the prediction could reasonably be made that if a particular strain had developed immunity to the post-translationally modified form of the protein, such immunity might not transfer to the precursor peptide. The producer bacterium, on the other hand, must at one time have had immunity to the precursor peptide assuming that the post-translationally modified form evolved over time from the precursor peptide.
Post-translational modification of native LL-2 may involve three or more enzymes, as was recently proposed for epidermin (Kupke, T., Stevanovic, S., Sahl, H-G, and Gotz, F., J. Bacteriol. 174 5354-5361 (1992)). Epidermin contains two lanthionine bridged rings and one methyl-lanthionine bridged ring (Schnell, N., Entian, K-D., Schneider, U., Gotz, F., Zahner, H., Kellner, R., and Jung, G, Nature 333 276-278 (1988)) similar to the modifications present in mature LL-2.
Published studies have communicated a belief that post-translational modifications are necessary for the activity of nisin-like proteins. This is no more apparent than in the AFRC (Agricultural Food Research Center, Reading, Berkshire, England) yearly report of 1991. The authors used a "gene probe designed from the predicted amino acid sequence of prenisin to clone the nisin biosynthetic region of a Lactococcus lactis nisin producer." Instead of using this sequence to produce the nisin peptide precursor directly, they created a system to mature and express the nisin precursor peptide and variants by inactivating the nisA nisin coding gene. Then the authors introduced a plasmid carrying the nisA gene or a variant to this system in order to study the mature protein and variants with novel properties. What the authors failed to investigate was the prenisin gene product itself for anti-bacterial activity. Perhaps the nisin secretion mechanism is inoperable for the precursor peptide sequences. It would be to the advantage of the bacterial strain, if the strain were extensively involved in post-translationally modifying nisin for the export mechanism not to recognize and secrete the unmodified precursor.
Morris, et al. (Morris, S. L., Walsh, R. C., and Hansen, J. N., Biol. Chem. 259, 13590-13594, (1984)) state "The activity of nisin is associated with dehydroalanine residues which could react with sulfhydryl groups". The twin observations were that nisin produces (1) inhibition of spore outgrowth and (2) inactivation of membrane sulfhydryl groups of germinated Bacillus cereus spores. The likelihood that nisin has "evolved the specific capability to inactivate membrane sulfhydryl groups of this (germinated B. cereus spores) type" is compared to the high specificity of "natural" antibiotic action.
Hansen (WO 90/00558 to Norman J. Hansen, 30 Jun., 1989) cloned the subtilin precursor gene into M13 along with a promotor (TATAAT) and ribosomal binding site (RBS). Similarly a nisin precursor sequence was cloned into M13mp18 for sequencing. In both cases, presence of a particular leader sequence between the RBS and the peptide sequence was claimed necessary to direct post-translational modification. Expression of the unmodified precursor peptide is discussed (hypothetically) in terms of excising the particular leader sequence responsible for post-translational modification. No use of the precursor protein is disclosed. In fact, whether the hypothetical experiment would work at all depends on the factors discussed previously including immunity to the precursor peptide, and detection of anti-bacterial activity.
The precursor peptide to nisin and LL-2 both contain a 23 amino acid residue leader region in addition to the 34 amino acid structural region (Buchman, G. W., Banerjee, S., and Hansen, J. N., J. Biol. Chem. 263 16260-16266, (1988)). The nisin gene is part of a polycistronic operon which may also contain genes for post-translational processing of the precursor peptide (Steen, M. T., Chung, Y. G., and Hansen, J. N., Appl. Environ. Microbiol. 57, 1181-1188, (1991)). The leader region and amino terminal end of nisin have considerable homology to epidermin (Kaletta, C. and Entian, K-D., J. Bacterio. 171, 1597-1601 (1989)). The leader sequence cleavage point (proline-arginine isoleucine) is the same in both precursor peptides. The first 5 member ring and the second 4 member ring are nearly identical except that Ile.sup.4, Dha.sup.5 and Leu.sup.6 in nisin are Lys.sup.4, Phe.sup.5 and Ile.sup.6 in epidermin. If Dha.sup.5 is a nucleophile important to biological activity, then Phe.sup.5 must play this role in epidermin. The C-terminal ends of epidermin and nisin are very different, showing no significant homology and having different numbers and types of bridges.
The DNA sequence of LL-2 differs from nisin in that the codon for the 27th amino acid, His is changed to Asn by natural mutation. The native protein encoded with this mutation (with post-translational modification and containing the 27 Asn mutation) has been described. (Mulders, J. W. M., Boerrigter, I. J., Rollema, H. S., Siezen, R. J., and de Vos, W. M., Eur. J. Biochem. 201, 581-584, (1991)). The DNA sequence was used to determine the protein sequence since only small parts of the protein sequence were determinable directly due to post-translational modification.
The structure of nisin had been determined prior to obtaining the gene sequence (Barber, M., et al., Experientia 44 266-270 (1988)), but a single ambiguity remained. The sole lanthionine bridge between residues 3 and 7 could have been formed by a cysteine either preceding a serine or following a serine in the DNA sequence. Prediction of an unambiguous precursor peptide from this data is not possible. By contrast, occurrences of methyl-lanthionine are unambiguously determinable since the portion of the molecule with the methyl group arises from the threonine portion of a cysteine, threonine pair.
Liu and Hansen (Appl. Environ. Microbiol. 56, 2551-2558 (1990)) used mercaptans or elevated pH to inactivate nisin. The cause of inactivation was presumed to be a chemical reaction between nucleophiles and dehydro residues, implying that dehydro residues are required for activity. The proposed chemical reaction was that the dehydro residues of nisin act as electrophilic Michael acceptors toward nucleophiles in the target. Treatment with cyanogen bromide resulted in two products. Each purified fragment was active but at a 10 fold diminished specific activity. The biological activity measured was the inhibition of B. cereus T spores in the elongated state. This measure of activity may not correlate well with other measurements of activity such as bacteriocidal activity.
Chan et al (Chan, W. C., Bycroft, B. W., Lian, L-Y, and Roberts, G. C. K., FEBS Letters 252, 29-36 (1989)) report an inactive degradation product in which dehydroalanine.sup.33, lysine.sup.34 and dehydroalanine.sup.5 (DHA.sup.5) are missing. The degradative removal of DHA.sup.5 causes an opening of the first ring structure. The loss of dehydroalanine.sup.33 and lysine.sup.34 alone (nisin.sup.1-32) did not cause inactivation. The biological activity discussed in this reference is measured by minimum inhibitory concentration (MIC, .mu.g/ml) against several gram positive and a single gram negative bacterium. Subsequent NMR studies (Lian, L-Y., Chan, W. C., Morley, S. D., Roberts, G. C. K., Bycroft, B. W., and Jackson, D., Biochem. J. 283 413-420, (1992)) of the same degradation product showed increased flexibility and lack of well defined structure in the region of DHA.sup.5 compared to nisin. This was interpreted as an indication of increased conformational flexibility because of removal of DHA.sup.5 and opening of the lanthionine defined ring. The hypothesis was drawn that dehydro residues in nisin play an important part in the mechanism of nisin antibiotic action. Dehydro residues were hypothesized to provide antibiotic action by reacting with a specific cellular nucleophile target.